The present invention relates to methods of treating fungal infections. More particularly, the present invention relates to methods of treating yeast infections with compounds that selectively target the NAD synthetase enzyme of yeast, with little or no attendant targeting of the NAD synthetase enzyme of the host.
The incidence of serious fungal infections, either systemic or topical, continues to increase for plants, animals, and humans. Fungi are plant-like eukaryotes that grow in colonies of single cells, called yeasts, or in filamentous mutlicellular aggregates, called molds. While many fungi are common in the environment and not harmful to plants or mammals, some are parasites of terrestrial plants and others can produce disease in humans and animals. When present in humans, mycotic (fungal) diseases can include contagious skin and hair infections, noncontagious systemic infections, and noncontagious foodborne toxemias. The incidence of such infections is not insignificant; in the U.S. approximately 10% of the population suffers from contagious skin and hair infections. While few healthy persons develop life-threatening systemic fungal infections, immunocompromised individuals, such as found in pregnancy, congenital thymic defects, or acquired immune deficiency syndrome (AIDS), can become seriously ill. This is further illustrated by the fact that fungal infections have become a major cause of death in organ transplant recipients and cancer patients.1 
Numerous antifungal agents have been developed for topical use against nonsystemic fungal infections. However, the treatment of systemic fungal infections, particularly in immunocrompromised hosts, continues to be a major objective in infectious disease chemotherapy. The organisms most commonly implicated in systemic infections include Candida spp., Cryptococcus neoformans, and Aspergillus spp., although there are a number of emerging pathogens. The major classes of systemic drugs in use currently are the polyenes (e.g., amphotericin B) and the azoles (e.g., fluconazole). While somewhat effective in otherwise healthy patients, these agents are inadequate in severely immunocompromised individuals. Furthermore, drug resistance has become a serious problem, rendering these antifungal agents ineffective in some individuals.2,3 
One reason for the limited number of systemic antifungal agents relates to the fact that, unlike bacteria, which are prokaryotes, yeast and molds are eukaryotes. Thus the biochemical make-up of yeast and molds more closely resembles eukaryotic human and animal cells. In general, this has made it difficult to develop antifungal drugs which selectively target in yeast an essential enzyme or biochemical pathway that has a close analog in humans and animals.
The ability to selectively inhibit the yeast form of a biochemical target with minimal effect on the mammalian form would provide a number of new approaches to the development of systemic antifungal drugs. In a few cases, this type of approach has already been proven to provide clinically useful systemic antifungal agents. For example, the mechanism of action for fluconazole, a widely used systemic antifungal drug, involves inhibition of a fungal C-14 demethylase, a cytochrome P450 enzyme that is essential for the production of the principal fungal sterol ergosterol. Ergosterol is very similar to the mammalian steroid cholesterol, and there is a closely related mammalian C-14 demethylase enzyme for which fluconazole is a much poorer inhibitor. This selectivity for inhibition of the fungal form of the enzyme over the mammalian form has resulted in the clinical utility of fluconazole.4 In a further example, preclinical studies on new antifungal agents that select for the yeast form over the mammalian form of a biochemical target include development of inhibitors for the plasma membrane ATPase5 and for topoisomerase I.6 
The inventors herein previously were part of a group that developed a number of antibacterial and antimicrobial agents that were targeted to NAD synthetase, an essential enzyme found in nearly all prokaryotic and eukaryotic cells. This enzyme is essential for the biosynthesis of nicotinamide adenine dinucleotide (NAD+), an essential cofactor in numerous enzymatic reactions. NAD synthetase catalyzes the last step in both the de novo and salvage pathways for NAD+ biosynthesis, which involves the transfer of ammonia to the carboxylate of nicotinic acid adenine dinucleotide (NaAD) in the presence of ATP and Mg+2. The overall reaction is illustrated in Scheme 1. 
Prokaryotic NAD synthetase is an ammonia-dependent amidotransferase that belongs to a family of xe2x80x9cN-typexe2x80x9d ATP pyrophosphatases; this family also includes asparagine synthetase and argininosuccinate synthetase.7 Unlike eukaryotic NAD synthetase found in yeast and mammals that can use glutamine as a source of nitrogen, the prokaryotic NAD synthetase of bacteria requires ammonia as the only nitrogen source. Furthermore, B. subtilis NAD synthetase, which was previously crystallized and used for drug design by the inventors, is a dimer with molecular weight around 65,000, while the yeast enzyme is multimeric and has at least 10 times larger molecular weight.8 These differences between eukaryotic and prokaryotic forms of NAD synthetase enzyme suggested that drugs specific for the prokaryotic enzyme could be designed, and the inventors subsequently developed inhibitors of this enzyme that are effective antibacterial and antimicrobial agents.9 Given these marked differences between prokaryotic and eukaryotic NAD synthetase, the inventors fully expected that the compounds would be selective for the prokaryotic NAD synthetase and would show little to no effect on eukaryotic NAD synthetase.
The present invention is based in part on the surprising discovery that NAD synthetase inhibitors are highly effective in inhibiting the growth of yeast, yet exhibited only moderate toxicity in animals. Thus, the present invention includes the use of NAD synthetase inhibitors as new antifungal agents for preventing or controlling parasitic yeast and mold infections in plants, and for the prophylactic or therapeutic treatment, topically and systemically, of fungal infections in humans and animals.
In a major aspect, the present invention provides a method of treating or preventing an antifungal infection in a host comprising administering to a host a treatment effective or treatment preventive amount of a yeast NAD synthetase enzyme inhibitor compound.
In a further aspect, the method of killing yeast with an amount of yeast NAD synthetase enzyme inhibitor to reduce or eliminate the production of NAD whereby the yeast is killed.
In yet another aspect, the invention provides a method of decreasing yeast growth, comprising contacting the yeast with an amount of a yeast NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD whereby yeast growth is decreased.
Additional advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.